Liposomes are vesicles composed of one or more lipid bilayers completely surrounding an internal aqueous space. They are usually made up of phospholipids or other amphipathic molecules either in pure form or in combination with other molecules such as sterols, long chain acids or bases, or membrane proteins. Liposomal structures vary from large (0.5 to 5 micron) multilamellar vesicles to small (250-750 angstrom) unilamellar vesicles. By convention, liposomes are categorized by size and a three letter acronym is used to designate the type of liposome being discussed. Multilamellar vesicles are generally designated (MLV). Small unilamellar vesicles are designated (SUV) and unilamellar vesicles are designated (LUV). In each case, the chemical composition is generally given following the acronym. See: D. Papahadjopoulos, Ann. N.Y. Acad. Sci., 308 1 (1978) and Ann. Rpts. Med. Chem., 14 250 (1979) the disclosures of which are incorporated herein by reference.
Liposomal preparations have been made by a number of techniques, including: ethanol injection, (Batzri et al., Biochim. Biophys. Acta, 298 1015 (1973)); ether infusion, (Deamer et al., Biochim. Biophys. Acta, 443 629 (1976) and Schieren et al., Biochim. Biophys. Acta, 542 137 (1978)), detergent removal, (Razin, Biochim. Biophys. Acta, 265 241 (1972)), solvent evaporation, (Matsumato et al., J. Colloid Interface Sci., 62 149 (1977)), evaporation of water in oil (REV) emulsions, (Szoka Jr. et al., Proc. Natl. Acad. Sci. USA, 75 4194 (1978)) and extrusions of MLV or LUV through a nucleopore polycarbonate membrane (Olson et al., Biochim. Biophys. Acta, 557 9 (1979)).
Liposomes may be used to affect cell behavior in vitro and in vivo. Magee and Miller (Nature, 235 339 (1972)) first reported that liposomes carrying antiviral antibody could protect cells against viral infection. Similar observations regarding liposomal protection of cells were noted by Gregoriadis and Buckland (Nature, 244 170 (1973)) who found that liposomes containing invertase could cause the disappearance of vacuoles of stored sucrose in mouse peritoneal macrophages. Papahadjopoulos and coworkers (Biochim. Biophys. Acta, 323 23 (1973)) reported that liposomes could induce cell fusion without cytotoxic effects.
Liposomes have been used to effect the cellular uptake of impermeant molecules, i.e., molecules that are not normally taken up. This action makes liposomes useful as carriers of foreign matter, such as drugs. For example, cyclic AMP inhibition of 3T3 cells growth in vitro was enhanced by 1000 fold using liposomes as the carrier (Papahadjopoulos et al., Nature, 252 163 (1974) and Papahadjopoulos et al., Biochim. Biophys. Acta, 363 404 (1974)). A similar increase in effectiveness was reported with actinomycin D in liposomes against a hamster cell line otherwise resistant to the drug (Papahadjopoulos et al., Cancer Res., 36 4406 (1976)).
Membrane fusion under moderately acidic conditions is responsible for the infection of a number of the enveloped viruses, including Semliki Forest virus (Marsh, et al., Cold Spring Harbor Symp. Quant. Biol., 46 835 (1982) and White et al., J. Cell Biol. 89 674 (1981)) vesicular stomatitis virus and influenza virus (White et al., supra). The precise mechanism of the acid induced membrane fusion is not known. Studies with liposome membranes have revealed that liposomes made of azolectin can fuse with the mitochondria inner membranes at pH 6.5 (Schneider et al., Natl. Acad. Sci. USA, 77 442-446 (1980)). Serum albumin (Schenkman et al., Chem. Phys. Lipids, 649 633 (1981) and Schenkman et al., Chem. Phys. Lipids, 28 165 (1981)) and its protelytic fragments (Chaimovich et al., Biophys. J., 41 28a (1983)) can induce liposome fusion at a pH below 4. Blumenthal et al. have shown that clathrin induces the fusion of neutral liposomes at a pH below 6.5 (Blumenthal et al., Biol. Chem., 258 3409 (1983)). In all these cases, liposome fusion requires the presence of some protein or other macromolecule.
Allens et al., (J. Cell Biol., 97 10) (a), Abstr. No. 419 (1983)) reported liposomes containing phosphatidylethanolamine and cholesterylhemisuccinate which are sensitive to pH in the endosomal pH region. These liposomes fuse with the endosomal membrane upon acidification of the endosome and thus deliver their contents to the cytoplasm.
Straubinger and coworkers (J. Cell Biol., 97 109(a), Abstr. No. 420 (1983)) reported the preparation of liposomes which became unstable at mildly acidic pH. These liposomes, which were composed of oleic acid, phosphatidylethanolamine and cholesterol (3:7:3 mol ratio), became permeable to the anionic fluorescent dye, calcein, below pH 7.0. These liposomes promoted the delivery of entrapped calcein to the cytoplasm of CV-1 cells.